Click-crosslinked hydrogels and methods of use

ABSTRACT

The present disclosure provides click-crosslinked hydrogels and methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national stage filing ofInternational Application No. PCT/US2015/024523, filed on Apr. 6, 2015,which in turn claims the benefit of priority of U.S. ProvisionalApplication No. 61/975,375, filed on Apr. 4, 2014, the disclosure ofeach of which is incorporated by reference in its entirety to the extentnot inconsistent with the disclosure herein.

GOVERNMENT SUPPORT

The invention was made with Government support under R01 HL069957awarded by the National Institutes of Health and under W911NF-13-1-0242awarded by the US Army Research Office. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

A hydrogel is a polymer gel comprising a network of crosslinked polymerchains. The network structure of hydrogels allows them to absorbsignificant amounts of water. Some hydrogels are highly stretchable andelastic; others are viscoelastic. Uses of hydrogels include celldelivery vehicles, cancer vaccines, tissue engineering (e.g., asscaffolding), and protein/small molecule delivery vehicles. Hydrogelsare also useful as structural materials. Some existing hydrogels areformed by crosslinking polymers via a noncovalent bond, such as an ionicbond, e.g., through calcium crosslinking Hydrogels created bynoncovalent bonds may not be as tough or stable long term as hydrogelscreated by covalent bonds. However, the existing chemistry used tocreate hydrogels with polymers linked by covalent bonds is complex andcostly. The chemical reactions are difficult to control with respect tothe reaction side reactions, cross reactivity, and obtaining hydrogelswith the desired physicochemical/mechanical properties, such aselasticity, strength, swelling extent, and degradation rate. Some of theexisting hydrogels also exhibit cytotoxicity and/or the polymerizationand crosslinking of the hydrogel cannot be created in the presence ofliving cells without causing significant cell death.

SUMMARY OF THE INVENTION

We recognized a need for covalent crosslinked hydrogels that are capableof being created using finely tunable and simple chemical reactions thatare nontoxic to cells, that can occur at a rapid rate under biologicalconditions, that are more stretchable and/or stronger than currentlyexisting hydrogels, and that are capable of being produced in acost-effective way. Our invention addresses these needs. In broadaspect, this disclosure provides novel bioorthogonal pair of functionalgroups that react via click chemistry to form crosslinkers that can beused to form biocompatible hydrogels.

Thus, in one aspect, the disclosure provides a hydrogel comprising afirst polymer and a second polymer, where the first polymer is connectedto the second polymer by linkers of formula (A):

wherein

-   -   bond        is a single or a double bond;    -   R¹ is —C₀-C₆alkyl-NR^(2N)—, —C₀-C₆alkyl —O—, or        —C₀-C₃alkyl-C(O)—;    -   R² is a bond, aryl, or heteroaryl, wherein aryl and heteroaryl        are optionally substituted with halogen, hydroxy, C₁-C₆alkyl,        C₁-C₆alkoxy, (C₁-C₆alkyl)amino, or di(C₁-C₆alkyl)amino;    -   R³ is —C₀-C₆alkyl-NR^(2N)—, —C₀-C₆alkyl-O—, or        —C₀-C₃alkyl-C(O)—; and    -   R⁴ is hydrogen, C₁-C₆alkyl, aryl, or heteroaryl, wherein aryl        and heteroaryl are optionally substituted with halogen, hydroxy,        C₁-C₆alkyl, C₁-C₆alkoxy, (C₁-C₆alkyl)amino, or        di(C₁-C₆alkyl)amino.

In one embodiment, the hydrogel of the disclosure is wherein the linkersof formula (A) are of the form of formula (I):

or by formula (II):

or by formula (III):

wherein the linkers of formula (I), (II), or (III) are optionallysubstituted at any suitable position.

In one embodiment, the linkers of formula (A) are wherein bond

is a single bond. In another embodiment, bond

is a double bond.

Another embodiment provides the linkers of formula (A) according to anypreceding embodiment, wherein R¹ is

-   -   a. —NR^(2N)—, —C₁-C₆ alkyl-NR^(2N)—, —O—, —C₁-C₆ alkyl —O—,        —C(O)—, or —C₁-C₃alkyl-C(O)—;    -   b. —C₀-C₆ alkyl-NR^(2N)—;    -   c. —C₁-C₆ alkyl-NR^(2N)—;    -   d. —C₁-C₃ alkyl-NR^(2N)—;    -   e. -methyl-NH— or -pentyl-NH—;    -   f. —C₀-C₆ alkyl-O—;    -   g. —C₁-C₆ alkyl-O—;    -   h. —C₁-C₃ alkyl-O—;    -   i. -methyl-O— or -pentyl-O—;    -   j. —C₀-C₃ alkyl-C(O)—;    -   k. —C(O)—;    -   l. -methyl-C(O)—;    -   m. the same as R³.

Another embodiment provides the linkers of formula (A) according to anypreceding embodiment, wherein R² is a bond.

In one embodiment, the linkers of formula (A) according to any precedingembodiment are those wherein R² is

-   -   a. aryl or heteroaryl, each optionally substituted;    -   b. optionally substituted aryl;    -   c. phenyl;    -   d. optionally substituted heteroaryl; or    -   e. pyridyl, pyrimidyl, or pyrazinyl.

Another embodiment provides the linkers of formula (A) according to anypreceding embodiment, wherein R³ is

-   -   a. —NR^(2N)—, —C₁-C₆ alkyl-NR^(2N)—, —O—, —C₁-C₆ alkyl —O—,        —C(O)—, or —C₁-C₃alkyl-C(O)—;    -   b. —C₀-C₆ alkyl-NR^(2N)—;    -   c. —C₁-C₆ alkyl-NR^(2N)—;    -   d. —C₁-C₃ alkyl-NR^(2N)—;    -   e. -methyl-NH— or -pentyl-NH—;    -   f. —C₀-C₆ alkyl-O—;    -   g. —C₁-C₆ alkyl-O—;    -   h. —C₁-C₃ alkyl-O—;    -   i. -methyl-O— or -pentyl-O—;    -   j. —C₀-C₃ alkyl-C(O)—;    -   k. —C(O)—;    -   l. -methyl-C(O)—; or    -   m. the same as R¹.

In one embodiment, the linkers of formula (A) according to any precedingembodiment are those wherein R⁴ is hydrogen.

In one embodiment, the linkers of formula (A) according to any precedingembodiment are those wherein R⁴ is

-   -   a. C₁-C₆ alkyl, aryl, or heteroaryl, wherein aryl and heteroaryl        are optionally substituted;    -   b. aryl or heteroaryl, wherein aryl and heteroaryl are        optionally substituted;    -   c. optionally substituted aryl;    -   d. phenyl;    -   e. optionally substituted heteroaryl; or    -   f. pyridyl, pyrimidyl, or pyrazinyl.

Another embodiment provides the linkers of formula (A) according to anypreceding embodiment, wherein R⁴ is C₁-C₆ alkyl, C₁-C₃ alkyl, or methyl.

In some embodiments, the hydrogel comprises a plurality of linkers offormula (A); or formula (I), formula (II), or formula (III).

The invention also includes a hydrogel comprising an interconnectednetwork of a plurality of polymers, e.g., including a first polymer anda second polymer. For example, the polymers are connected via aplurality of linkers of formula (A), or of formula (I), formula (II), orformula (III).

Some embodiments of the disclosure provide hydrogels wherein the firstpolymer and the second polymer are independently soluble polymers. Inother embodiments, the first polymer and the second polymer areindependently water-soluble polymers.

In some cases, the concentration of crosslinks per hydrogel (e.g., whereeach crosslink comprises formula I) is at least about 10% (w/w), e.g.,at least about 10%, about 15%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 97%,about 99%, or about 100% (w/w).

The first polymer and the second polymer can be the same or different.In some embodiments, the first polymer and the second polymer are thesame type of polymer. In other embodiments, the first polymer and/or thesecond polymer comprise a polysaccharide. For example, the first polymerand the second polymer can both comprise a polysaccharide. In someembodiments, the first polymer and/or the second polymer areindependently selected from the group consisting of alginate, chitosan,polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen,chondroitin, agarose, polyacrylamide, and heparin. In some embodiments,the first polymer and the second polymer are the same polymerindependently selected from the group consisting of alginate, chitosan,polyethylene glycol (PEG), gelatin, hyaluronic acid, collagen,chondroitin, agarose, polyacrylamide, and heparin.

Some embodiments of the disclosure provide hydrogels wherein the firstpolymer and the second polymer are independently selected from groupconsisting of alginate, chitosan, and gelatin. In one embodiment, thefirst polymer and the second polymer are independently alginate. Inanother embodiment, the first polymer and the second polymer areindependently chitosan. In another embodiment, the first polymer and thesecond polymer are independently gelatin.

In some embodiments, the first polymer and/or the second polymer ismodified by a cell adhesive peptide, e.g., an extracellular cell matrix(ECM) component. In one embodiment, the first and/or second polymerso-modified is alginate. The cell adhesive peptide can comprise, forexample, the amino acid sequence arginine-glycine-aspartate (RGD).Examples include the amino acid sequencearginine-glycine-aspartate-cysteine (RGDC) (SEQ ID NO: 1) andarginine-glycine-aspartate-serine (RGDS) (SEQ ID NO: 2). In otherexamples, the cell adhesive peptide comprises the amino acid sequence oflysine-glutamine-alanine-glycine-aspartate-valine (KQAGDV) (SEQ ID NO:3) or valine-alanine-proline-glycine (VAPG) (SEQ ID NO: 4). In someexamples, the cell adhesive peptide is CGGGGRGDSP (SEQ ID NO: 5). Othercell adhesive peptides may be used based on the desired application andwill be apparent to one of skill in the art.

In some cases, the cell adhesive peptide is covalently linked to thepolymer via a thiol-ene reaction, e.g., via thiol-ene photochemistry.For example, the cell adhesive peptide can be covalently linked to thepolymer (e.g., alginate) prior to or following crosslinking of thepolymers to form a hydrogel. Such use of thiol-ene reaction tocovalently like the cell adhesive peptide to the polymer issignificantly faster and more efficient than the previously disclosedmethods, such as methods using of carboxyl activating agents (e.g., EDC)to couple the peptide to the polymer.

In some examples, the hydrogel is elastic. For example, the Young'smodulus of the hydrogel of the disclosure can be about 50 to about50,000 Pa, e.g., about 50 to about 500 Pa, or about 50 to about 1,000Pa, or about 50 to about 5,000 Pa, or about 50 to about 10,000 Pa, orabout 500 to about 50,000 Pa, or about 500 to about 10,000 Pa, or about500 to about 5000 Pa, or about 500 to about 1,000 Pa, or about 1,000 toabout 50,000 Pa, or about 1,000 to about 10,000 Pa, or about 1,000 toabout 5,000 Pa, or about 50 to about 20,000 Pa, or about 500 to about20,000 Pa, or about 1,000 to about 20,000 Pa, or up to about 40,000 Pa,or up to about 30,000 Pa, or up to about 20,000 Pa.

In some embodiments, the hydrogel further comprises a cell, a biologicalfactor, and/or a small molecule.

Exemplary cells include myoblasts for muscle regeneration, repair orreplacement; hepatocytes for liver tissue regeneration, repair or organtransplantation, chondrocytes for cartilage replacement, regeneration orrepair, pancreatic islets (e.g., for treatment of diabetes), andosteoblasts for bone regeneration, replacement or repair, various stemcell populations (embryonic stem cells differentiated into various celltypes), bone marrow or adipose tissue derived adult stem cells, cardiacstem cells, pancreatic stem cells, endothelial progenitors and outgrowthendothelial cells, mesenchymal stem cells, hematopoietic stem cells,neural stem cells, satellite cells, side population cells,differentiated cell populations including osteoprogenitors andosteoblasts, chondrocytes, keratinocytes for skin, tenocytes for tendon,intestinal epithelial cells, endothelial cells, smooth muscle cells andfibroblasts for tissue or organ regeneration, repair or replacementand/or for DNA delivery. Preferably, the cells are mammalian, e.g.,human; however, the system is also adaptable to other eucaryotic animalcells, e.g., canine, feline, equine, bovine, and porcine as well asprokaryotic cells such as bacterial cells.

A “biological factor” is a molecule having some sort of in vitro or invivo biological activity. In some examples, the biological factor is aprotein (e.g., peptide, polypeptide, antibody, or fragment thereof),nucleic acid (e.g., DNA, RNA, modified DNA or RNA, or aptamer), lipid(e.g., phospholipid), or carbohydrate (e.g., polysaccharide orproteoglycan).

Some biological factors are capable of maintaining cell viability,promoting cell proliferation, or preventing premature terminaldifferentiation of cells. Such biological factors are used alone or incombination to achieve the desired result.

Biological factors suitable for use in the present invention include,but are not limited to: growth factors, hormones, neurotransmitters,neurotransmitter or growth factor receptors, interferons, interleukins,chemokines, cytokines, colony stimulating factors, chemotactic factors,MMP-sensitive substrate, extracellular matrix components; such as growthhormone, parathyroid hormone (PTH), bone morphogenetic protein (BMP),transforming growth factor-α (TGF-α), TGF-β1, TGF-β2, fibroblast growthfactor (FGF), granulocyte/macrophage colony stimulating factor (GMCSF),epidermal growth factor (EGF), platelet derived growth factor (PDGF),insulin-like growth factor (IGF), scatter factor/hepatocyte growthfactor (HGF), fibrin, collagen, fibronectin, vitronectin, hyaluronicacid, an RGD-containing peptide or polypeptide, an angiopoietin andvascular endothelial cell growth factor (VEGF). Splice variants of anyof the above mentioned proteins, and small molecule agonists orantagonists thereof that may be used advantageously to alter the localbalance of pro and anti-migration and differentiation signals are alsocontemplated herein.

Examples of cytokines as mentioned above include, but are not limited toIL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18,granulocyte-macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumornecrosis factor (TNF), TGF-β, FLT-3 ligand, and CD40 ligand.

Suitable biological factors useful in accordance with the invention alsoinclude but are not limited to DNA molecules, RNA molecules, antisensenucleic acids, ribozymes, plasmids, expression vectors, marker proteins,transcription or elongation factors, cell cycle control proteins,kinases, phosphatases, DNA repair proteins, oncogenes, tumorsuppressors, angiogenic proteins, anti-angiogenic proteins, cell surfacereceptors, accessory signaling molecules, transport proteins, enzymes,anti-bacterial agents, anti-viral agents, antigens, immunogens,apoptosis-inducing agents, anti-apoptosis agents, and cytotoxins.

The disclosure also features a method for preparing a hydrogelcomprising:

-   a) providing a first polymer comprising a tetrazine moiety and a    second polymer comprising at least one norbomene moiety. The number    of tetrazine moieties in the first polymer (denoted by ‘t’ in the    examples below) can be any integer between 1 and 100,000:

-   -   The number of norbornene moieties in the second polymer (denoted        by ‘n’ in the examples below) can be any integer between 1 and        100,000:

-   b) contacting the second polymer with the first polymer to form a    cross-linked polymer having crosslinks of formula (A). In one    embodiment, the crosslinks of formula (A) are of the form of formula    (I),

-   -   or formula (II),

-   -   or formula (III):

wherein the linkers of formula (I), (II), or (III) are optionallysubstituted at any suitable position.

In some cases, each molecule of the first polymer comprises about1-50,000 tetrazine moieties, e.g., about 1-10,000, about 1-5000, about1-1000, about 5000-50,000, about 5000-10,000, about 1000-10,000, about1000-5000, about 500-5000, about 500-1000, or about 1-500 tetrazinemoieties. In some cases, each molecule of the second polymer comprisesabout 1-50,000 norbornene moieties, e.g., about 1-10,000, about 1-5000,about 1-1000, about 5000-50,000, about 5000-10,000, about 1000-10,000,about 1000-5000, about 500-5000, about 500-1000, or about 1-500norbornene moieties. In some embodiments, step b) of the methodcomprises contacting a second polymer with a first polymer at a ratio ofabout 1:104 to about 10:1 (second polymer:first polymer). For example,the ratio of the second polymer to the first polymer is about 1:10, orabout 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, orabout 1:4, or about 1:3, or about 1:2, or about 1:1, or about 2:1, orabout 3:1, or about 4:1, or about 5:1, or about 6:1, or about 7:1, orabout 8:1, or about 9:1, or about 10:1.

In some cases, the tetrazine moiety is coupled to the polymer byreacting the polymer with benzyl amine tetrazine and a coupling agent.In other examples, the tetrazine moiety is coupled to the alginate byreacting the polymer with benzyl alcohol tetrazine or benzoic acidtetrazine and a coupling agent. Exemplary coupling agents include, butare not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),carbonyl diimidazole, N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC), andN,N,N′,N″-tetramethyl-O-(1H-benzotriazol-1-yl)uroniumhexafluorophosphate (HBTU).

In some cases, the norbornene moiety is coupled to the polymer byreacting the polymer with norbornene methanamine and a coupling agent.In other examples, the norbomene moiety is coupled to the polymer byincubating the polymer with norbornene methanol or norbornene carboxylicacid and a coupling agent. Exemplary coupling agents include, but arenot limited to, EDC, carbonyl diimidazole, DCC, DIC, and HBTU. In somecases, N-Hydroxysuccinimide (NHS) is also included in the couplingreaction.

In some embodiments, the crosslinking capacity of the polymers has amaximum limit based on the degree of substitution of functional groups(e.g., norbornene and tetrazine) on the polymer(s). The crosslinkingcapacity can be characterized and measured, for example, mechanically bydemonstrating that higher theoretical crosslinking density (e.g., thedegree of substitution or the ratio of the first polymer:the secondpolymer) results in a stiffer or more highly crosslinked network ofpolymers in the hydrogel.

In some embodiments of the method, the crosslinking reaction proceeds asa spontaneous chemical reaction, such as with no input of light, heat,radicals needed. The crosslinking reaction can occur in water, inaqueous buffers or cell culture media (e.g., phosphate buffered saline,Hank's balanced salt solution, Dulbecco's Modified Eagle Medium, and thelike), or in organic solvents (e.g., methanol, ethanol, dichloromethane,dimethylformamide, and the like). In addition, the crosslinking reactioncan occur at a wide range of temperatures, such as about −80° C. to atleast about 50° C., about −80° C., about −20° C., about 4° C., about 22°C., about 37° C., or about 50° C. In some examples, the crosslinkingreaction occurs at a usable range of temperature and conditions forforming hydrogels and occurs without the input of external energy.

In some embodiments of the method, after crosslinking, unreactednorbornene and/or tetrazine remain on the polymers. In some cases theunreacted norbornene and/or tetrazine functional groups on thecrosslinked hydrogel can be used for post-crosslinking reactionmodification of the gel. The amount of unreacted norbornene and/ortetrazine on the polymers can be modulated by varying the ratios of thefirst polymer to the second polymer used during the crosslinkingreaction.

The disclosure also features a method for preparing a hydrogelcomprising:

-   a) providing a first polymer comprising a diene moiety and a second    polymer comprising at least one dienophile moiety, wherein the diene    is selected from:

-   -   wherein R is a suitable spacer for linking to the first polymer,        and R⁴ is as defined above;        and the dienophile is selected from:

-   -   wherein R′ is a suitable spacer for linking to the second        polymer; and

-   b) contacting the second polymer with the first polymer to form a    cross-linked polymer.

In one embodiment, the disclosure provides a hydrogel prepared by theabove method, comprising a first polymer and a second polymer, whereinthe first polymer is connected to the second polymer by linkersresulting in Diels-Alder addition of a diene and dienophile moiety,wherein the diene is selected from:

-   -   wherein R is a suitable spacer for linking to the first polymer,        and R⁴ is as defined above;        and the dienophile is selected from:

-   -   wherein R′ is a suitable spacer for linking to the second        polymer.

In one embodiment, the hydrogel is wherein R is R¹-R² moiety, wherein R¹and R² are as defined above; and R′ is R³, wherein R³ is as definedabove.

The disclosure also provides a method of regenerating a tissue in asubject in need thereof comprising administering a hydrogel describedherein further comprising cells to the subject. For example, the cellscan be mammalian cells, and the tissue a mammalian tissue. In someembodiments, the mammalian cell is of the same type as the tissue to beregenerated. In other embodiments, the mammalian cell is a stem cell.Embodiments of the method include contacting a mammalian tissue with thehydrogel, where the hydrogel further comprises a first component thattemporally regulates egress of the cell from the hydrogel and/or asecond component that spatially regulates egress of the cell from thehydrogel.

In another example, a method of regenerating a target tissue of a mammalcomprises providing a hydrogel described herein, where the hydrogelcomprises a mammalian cell immobilized within the hydrogel (i.e., themammalian cell remains within the hydrogel for an extended period oftime without exiting the hydrogel). The method includes contacting amammalian tissue with the hydrogel, where the mammalian cell isimmobilized within the hydrogel, and where the hydrogel comprises afirst component that temporally regulates egress of a cell of themammalian cell from the hydrogel and a second component that spatiallyregulates egress of the mammalian cell from the hydrogel. In someembodiments the mammalian cell is a progeny cell. In some embodiments,the hydrogel remains stable and does not allow for host cellinfiltration.

In one embodiment, the hydrogel described herein is useful as animmunoprotective barrier, e.g., for pancreatic islet transplantation. Insome cases, pancreatic islet transplantation is a treatment fordiabetes, e.g., Type I diabetes. Transplanted cells such as islets canbe destroyed by immune reactions, and the hydrogels of the invention arecapable of encapsulating cells such as islet cells prior toimplantation/injection of the hydrogel. In this way, the hydrogels serveas an immunoprotective barrier in the body and minimize immune rejectionof transplanted cells and tissues.

Additionally, the disclosure provides a method of modulating an activityof a cell in a mammal, comprising

-   (a) administering to a mammal a hydrogel described herein, where the    hydrogel further comprises a recruitment composition incorporated    therein or thereon; and-   (b) contacting the cell with a deployment signal, wherein the    deployment signal induces egress of the cell, and wherein the    activity of the cell at egress differs from that prior to entering    the hydrogel.

For example, the cell can be an immune cell and can be a dendritic cell,macrophage, T cell, or B cell.

In other embodiments, the hydrogels described herein are useful forlong-term cell transplantation, i.e., encapsulated cells reside andproliferate in or on the hydrogel for at least 1 day (e.g., at least 1,2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12 months, or 1, 2, 3, 4 years or more). For example, lessthan about 50% (e.g., less than about 50%, about 40%, about 30%, about20%, about 10%, or less) of the encapsulated cells are released from thehydrogel into a surrounding tissue at least 1 day (e.g., at least 1, 2,3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 months, or 1, 2, 3, 4 years or more) after administrationof the hydrogel into a subject. In some cases, the hydrogels are capableof retaining the encapsulated cells in part due to the high level ofstability (in structure and susceptibility to degradation) of thehydrogels.

The hydrogels and methods disclosed herein provide certain advantages.The click-crosslinked hydrogels of the invention are stronger andtougher than other types of hydrogels, e.g., hydrogels that are createdvia ionic crosslinks or a combination of ionic crosslinks and covalentcrosslinks. The hydrogels of the disclosure are also robust and canmaintain structural integrity for months in vivo. In contrast, thecurrently used cell compatible materials require ionic crosslinking (dueto covalent bio-orthogonal issues given above), which does not maintainextended structural integrity. To date, there has been no disclosure ofother covalent crosslinking strategy (for example, in alginate, gelatin,chitosan at least) that has the level of biocompatibility and/orbioorthogonality of the hydrogels disclosed herein.

Also, the hydrogels of described herein can be generated by crosslinkingpolymers in the presence of cells and do not necessarily have to becrosslinked and formed prior to introduction of cells into the hydrogel.In addition, the hydrogels described herein can be generated bycrosslinking polymers in the presence of a wide range of biologicalfactors (e.g., morphogens, cytokines, and proteins) without damagingthese biological factors during the crosslinking process.

Additionally, the methods of the invention utilize click chemistrybetween norbomene and tetrazine functional groups, and this reaction hasbeen shown to be rapid (gelling within an hour) and allows for in situgelation of the disclosed biomaterials. Quite advantageously, the clickchemistry between norbomene and tetrazine functional groups of thedisclosure is employable and reliable in aqueous systems, such asbiologically compatible conditions (e.g., pH, temperature, and watercontent of biological systems) and can be applied to a wide variety ofbiomaterials. Further, the click chemistry between norbomene andtetrazine of the disclosure infers solubility to hydrogels atphysiological relevant pHs. For example, chitosan hydrogel is moresoluble at physiological pH, whereas chitosan polymer is insoluble inwater and requires solubilization in dilute acid, pH˜2.

The methods of the invention can utilize one type of polymer, whichprovides more control over the assembly of the hydrogel and improvedcontrol over the crosslinking process, such as by controlling thepercentage of tetrazine and norbornene side chains that are able toreact with each other to form a hydrogel with the desiredphysicochemical and mechanical properties. In contrast, prior artcovalent crosslinking strategies use biomaterials that can cross-reactwith cells or biological agents, or require polymerization initiatorsthat can damage these materials. Covalent crosslinking according to themethods disclosed herein can be accomplished in the presence of cellsand biological factors without altering or otherwise damaging them.

Also, the methods of the invention provide improved control overmechanical properties of the hydrogel (e.g., elasticity and strength,swelling ratios, and degradation profiles). These hydrogel mechanicalcharacteristics can be tuned through manipulation of the polymermolecular weight(s), degree of click substitution, and/or stoichiometryof the click chemistry components. Another advantage of the methods andhydrogels is the ability to utilize non-reacted functional groups on thepolymer(s) to covalently couple desired agents (e.g., cell adhesivepeptides) after the hydrogel has been synthesized. Yet another advantageis the ability of the polymers to be crosslinked in situ to form theclick cross-linked hydrogel without the need for external couplingagents or catalysts. This is especially advantageous for the formationand use of injectable hydrogels, i.e., hydrogels that are injectable asa flowable composition (e.g., a liquid) but that form a gel in the bodyafter injection. The conjugation approach of the disclosure expands theversatility of biomaterial hydrogels.

Additionally, some previously available gels are based on 4-armPEG-plus-peptide based systems. Our disclosure advantageously provides aone-component system (e.g., an alginate system or a chitosan system or agelatin system), which is simpler and cheaper than other systems such asthe PEG-peptide systems. This is in part because the polymer (e.g.,alginate, gelatin, or chitosan) is inexpensive and in some embodimentscan be the single base polymer for both of the two components of thehydrogels of the invention. Also, the disclosure provides the firstdemonstration of a tetrazine-norbornene click-chemistry crosslinkedpolysaccharide material. Moreover, the crosslinking reaction describedherein can take place at a wide range of temperatures and conditions,which provides a robustness that allows for the generation ofhydrogels/cryogels with similar and/or better performance than thosepreviously available, including, for example, the ability to encapsulatemore molecules and/or drugs in the gel than previously possible whilekeeping the cost and complexity to a minimum.

In the methods of the disclosure, norbornene can be efficiently(e.g., >95%) conjugated to thiol containing small molecules, polymers,peptides and proteins (through cysteines) in minutes via photoinitiatedradical reaction. By contrast, prior art methods require extendedreaction times (often overnight) with very low efficiency (e.g., lessthan 40%). The norbornene conjugate can be incorporated into gels in ahomogenous manner through the methods of the disclosure. Further,conjugation of amine, thiol, hydroxyl, or carboxylic acid containingmolecules (small molecules, peptides, proteins, etc.) can allow for thehomogenous covalent crosslinking of these molecules to the hydrogelsduring gelation for either permanent or hydrolyzable biomaterials.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing fabrication of click alginate hydrogels.In the figure aqueous carbodiimide chemistry is used to modify alginatebackbone carboxylic acids with tetrazine or norbornene, resulting inAlg-T or Alg-N polymers respectively.

FIG. 2 is a schematic showing a click alginate polymer cross-linkingreaction where Alg-T and Alg-N polymers are mixed together to create acovalently crosslinked click alginate hydrogel network, with the loss ofN₂.

FIG. 3A illustrates representative in situ dynamic rheometry plot at 25°C. for 3% w/v click alginate at N:T=1, demonstrating modulus evolutionwith time. FIG. 3B illustrates compressive Young's modulus, and FIG. 3Cillustrates volumetric swelling ratios for 2%, 3% and 4% w/v clickalginate hydrogels at varying N:T ratio. Values represent mean andstandard deviation (n=4).

FIGS. 4A-B illustrate 3T3 fibroblast encapsulation in click crosslinked(2% w/v click crosslinked (N:T=1)) and ionically crosslinked alginatehydrogels stained with ethidium homodimer-1 (red) for dead cells at 4hours and 3 days post encapsulation (scale bar=100 μm) (4A).Quantitative analysis of cell viability (Two-Way ANOVA with Sidak'spost-hoc test, **p<0.01, ***p<0.001; Values represent mean and standarddeviation, n=4) and overall metabolic activity as measured by reductionof AlamarBlue over time in culture (n=6) (4B).

FIGS. 5A-E illustrate cell adhesion, spreading, and proliferation onclick alginate hydrogels modified with RGD peptides after synthesis.Schematic of CGGGGRGDSP peptide coupling reaction onto click alginatehydrogel surface using photoinitiated thiol-ene chemistry (5A).Representative images of 3T3 fibroblast adhesion, spreading, andproliferation on click alginate hydrogels with varying RGD peptidedensity (scale bar=200 μm) (5B), and quantification (Two-Way ANOVA withTurkey's post-hoc test, *p<0.05, ****p<0.0001 relative to No RGDcontrol; Values represent mean and standard deviation, n=4-7) byendogenous EGFP expression (green) over 3 days (7C). Phalloidin (red)and Hoescht 33342 (blue) staining of F-actin filaments and nuclei at 3days for cells adherent to RGD modified click alginate hydrogels (scalebar=100 μm) (5D). Representative fluorescent images of EGFP (green) 3T3cells cultured on click alginate hydrogels with varying ligand densityfor 3 days and stained with ethidium homodimer-1 (red) (scale bar=100μm) (5E). The High, Low, and No RGD conditions refer to the 2 mM, 0.2mM, and 0 mM peptide solutions used to modify the click alginatehydrogel surface.

FIG. 6A illustrates tissue response following subcutaneous injection ofclick and ionically crosslinked hydrogels in vivo. Representativehematoxylin and eosin (H&E) stain of tissue sections at 1 week, 1 month,and 2 month following injection into BALB/cJ mice (scale bar=150 μm).Images focus on the gel-tissue interface, with dashed lines indicatingthe border between the hydrogel and the surrounding tissue. Asterisksindicate the location of the click alginate hydrogel, which separatesfrom the tissue during histological analysis with no cell infiltration.FIG. 6B focuses on interior of the hydrogel at 2 months followingsubcutaneous injection in vivo (scale bar=200 μm).

FIG. 7A is a schematic showing a click gelatin polymer cross-linkingreaction where Gel-Tz and Gel-Nb polymers are mixed together to create acovalently crosslinked click alginate hydrogel network, with the loss ofN₂. FIG. 7B (left) illustrates storage modulus (G′) time sweeps (onrheometer) of click gelatin hydrogels at 37° C. at 5 and 10% w/v andN:T=1. Plateau modulus reached within 45 min for both, with insetshowing time to 50% of plateau modulus. On the right, plateau storagemodulus is shown as a function of N:T polymer ratio, wherein mechanicalproperties are controlled by changing the ratio of polymers that arebeing mixed (rather than amount of crosslinker or concentration ofpolymer).

FIGS. 8A-B illustrate 3T3 fibroblast adhesion, spreading, andproliferation on click gelatin hydrogels.

FIG. 9 (top) shows that 3T3 fibroblasts retained high viability after 3Dencapsulation in click crosslinked gelatin with an increase in metabolicactivity over a three day culture period. Bottom figure shows thatencapsulated NIH/3T3 cells rapidly assume a spread morphology withencapsulation in click crosslinked gelatin after one day (cell length:80±6 μm), whereas cells treated with broad-spectrum matrixmetalloproteinase(MMP)-inhibitor marimastat do not assume a spreadmorphology (cell length: 16±2 μm).

DETAILED DESCRIPTION OF THE INVENTION

Before the disclosed methods and materials are described, it is to beunderstood that the aspects described herein are not limited to specificembodiments, apparatuses, or configurations, and as such can, therefore,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and, unlessspecifically defined herein, is not intended to be limiting.

Throughout this specification, unless the context requires otherwise,the word “comprise” and “include” and variations (e.g., “comprises,”“comprising,” “includes,” “including”) will be understood to imply theinclusion of a stated component, feature, element, or step or group ofcomponents, features, elements or steps but not the exclusion of anyother integer or step or group of integers or steps.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context dictatesotherwise.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. Quantitative values described herein using the modifier“about” can include ranges of ±10% of the stated value.

As used herein, the term “aryl” represents an aromatic ring systemhaving a single ring (e.g., phenyl) which is optionally fused to otheraromatic hydrocarbon rings or non-aromatic hydrocarbon rings. “Aryl”includes ring systems having multiple condensed rings and in which atleast one is carbocyclic and aromatic, (e.g.,1,2,3,4-tetrahydronaphthyl, naphthyl). Examples of aryl groups includephenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl, dihydronaphthyl,fluorenyl, tetralinyl, and 6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl.In certain examples, aryl groups include those having a firstcarbocyclic, aromatic ring fused to an aromatic or aliphaticheterocycle, for example, 2,3-dihydrobenzofuranyl. The aryl groupsherein are unsubstituted or, when specified as “optionally substituted”.

As used herein, the term “alkyl” means a saturated hydrocarbon having adesigned number of carbon atoms, such as 1 to 6 carbons (i.e., inclusiveof 1 and 6), 1 to 6 carbons, 1 to 3 carbons, or 1, 2, 3, 4, 5 or 6.Alkyl group may be straight or branched and depending on context, may bea monovalent radical or a divalent radical (i.e., an alkylene group). Inthe case of an alkyl or alkyl group having zero carbon atoms (i.e.,“C₀alkyl”), the group is simply a single covalent bond if it is adivalent radical or is a hydrogen atom if it is a monovalent radical.For example, the moiety “—(C₀-C₆alkyl)-O—” signifies connection of anoxygen through a single bond or an alkylene bridge having from 1 to 6carbons and C₀-C₃alkyl represents a bond, methyl, ethyl, and propylmoieties. Examples of “alkyl” include, for example, methyl, ethyl,propyl, isopropyl, butyl, iso-, sec- and tert-butyl, pentyl, and hexyl.

The term “alkoxy” represents an alkyl group of indicated number ofcarbon atoms attached to the parent molecular moiety through an oxygenbridge. Examples of “alkoxy” include, for example, methoxy, ethoxy,propoxy, and isopropoxy.

The terms “halogen” or “halo” indicate fluorine, chlorine, bromine, andiodine.

As used herein, the term “heteroaryl” refers to an aromatic mono- orbi-cyclic ring system of 3-14 atoms ring system containing at least oneheteroatom selected from nitrogen, oxygen and sulfur in an aromaticring. Most commonly, the heteroaryl groups will have 1, 2, 3, or 4heteroatoms. The heteroaryl may be fused to one or more non-aromaticrings, for example, cycloalkyl or heterocycloalkyl rings. In oneembodiment of the present compounds the heteroaryl group is bonded tothe remainder of the structure through an atom in a heteroaryl grouparomatic ring. In another embodiment, the heteroaryl group is bonded tothe remainder of the structure through a non-aromatic ring atom.Examples of heteroaryl groups include, for example, pyridyl,pyrimidinyl, quinolinyl, benzothienyl, indolyl, indolinyl, pyridazinyl,pyrazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl,phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl,indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl,furanyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl,benzo[1,4]oxazinyl, triazolyl, tetrazolyl, isothiazolyl, naphthyridinyl,isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl,isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl, isobenzothienyl,benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl,benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, pteridinyl,benzothiazolyl, imidazopyridinyl, imidazothiazolyl,dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl,dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, chromonyl,chromanonyl, pyridinyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl,dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl,dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl,pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinylN-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N-oxide,isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide,phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolylN-oxide, thiazolyl N-oxide, indolizinyl N-oxide, indazolyl N-oxide,benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide,oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolylN-oxide, benzothiopyranyl S-oxide, benzothiopyranyl S,S-dioxide.Preferred heteroaryl groups include pyridyl, pyrimidyl, pyrazinyl,quinolinyl, indolyl, pyrrolyl, furanyl, thienyl, imidazolyl, pyrazolyl,indazolyl, thiazolyl and benzothiazolyl. The heteroaryl groups hereinare unsubstituted or, when specified as “optionally substituted”.

As used herein the term “contacting” includes the physical contact of atleast one substance to another substance, either directly or indirectly.An example of indirect contacting is injecting a hydrogel into a mammalto result in the hydrogel contacting a tissue.

As used herein the term “sufficient amount” and “sufficient time”includes an amount and time needed to achieve the desired result orresults, such as to dissolve a portion of the polymer.

All percentages, ratios and proportions herein are by weight, unlessotherwise specified. A weight percent (weight %, also as wt %) of acomponent, unless specifically stated to the contrary, is based on thetotal weight of the formulation or composition in which the component isincluded (e.g., on the total amount of the active material).

All temperatures are in degrees Celsius (° C.) unless otherwisespecified.

In view of the present disclosure, the methods and compositionsdescribed herein can be configured by the person of ordinary skill inthe art to achieve a desired goal. In general, the disclosedcompositions and methods provide several unmet advantages. For example,the compositions and methods described herein provide a new method formaking polysaccharide hydrogels using bioorthogonal chemistry. As theclick alginate polymers and hydrogels are biocompatible and provide abioorthogonal cross-linking reaction, there are many potentialapplications of these modified biopolymers, ranging from in vitro cell,protein, and drug encapsulation and release, to in vivo hydrogels withlong-term stability. Though previous polymer systems may be capable ofsome of these applications, click polymers of the disclosure provideadditional capabilities outside of canonical polymer hydrogels that wereprevious unattainable due to incompatible chemistries or contaminants.For instance, the hydrogels provided by the disclosure allow for thestudy of biological behavior of cells in 3 dimensions (3D)s, whichprovides a platform for observing spreading, proliferation,differentiation in a material system that does not interfere with thecells. Additionally, the polymers/hydrogels of the disclosure can beused in the field of super tough hydrogels that had been generatedpreviously using a combination of ionic and click-crosslinked alginatepolymers. See, e.g., Sun, et al. (2013). Highly stretchable and toughhydrogels. Nature, 489(7414), 133-136. The methods of the disclosurecreate a tougher alginate hydrogel compared to previous hydrogels, suchas ionically crosslinked or ionic plus click-crosslinked hydrogels.Other engineering applications such as 3D cell/structure printing canalso be developed with this system. For example, 3D printing can beaccomplished by using the click alginate polymers as the “inks” thatsolidy and/or form structures once mixed and printed out of the printer.In some examples, during the printing process, cells and/or biologicalfactors/small molecules can also be encapsulated within the network ofpolymers to form the hydrogel. Click alginate polymers are also useful,e.g., alone, as bulking agents. For example, the hydrogels of thedisclosure can be applied (e.g., not in combination with other agents)for cosmetic and aesthetic applications, or in other situations wheretissue bulking is needed for function such as incontinence treatment.For example, cosmetic uses include the treatment of wrinkles, thefilling of small tissue defects with the hydrogels described herein,e.g., alginate gels in addition to or comprising fat forming cells (see,e.g., US 2013-0195764; WO 12/148684; WO 12/167230; and WO 12/167230,incorporated herein by reference). In some examples, the hydrogels ofthe disclosure are useful for tissue bulking for gastroesophageal refluxdisease (GERD), incontinence treatment, and/or urinary reflux. In somecases, hydrogels are injected into the appropriate tissue site, wherethe hydrogel would provide expansion of the tissue and long-termmaintenance of this expanded volume, in part due to the high level ofstability of the hydrogel.

There are currently methods to generate polyethylene glycol (PEG)-basedhydrogels. See, e.g., Alge, et al. (2013). Synthetically Tractable ClickHydrogels for Three-Dimensional Cell Culture Formed UsingTetrazine-Norbomene Chemistry. Biomacromolecules, 14(4), 949-953.However, while these PEG-based hydrogels are capable of encapsulatingcells and patterning, this system has not been developed into anyapplications. Unlike the instant disclosure, to make the PEG-basedhydrogels, Alge grafts tetrazine onto the PEG polymers, and then use acleavable peptide bearing norbornene on each end to crosslink thosepolymers. In contrast, the click polymer system of the disclosure is atwo-component system where the base polymer is the same, but has beenmodified differently to form the two components. This click polymersystem of the disclosure is beneficial for significantly decreasingcomplexity and cost of the material system.

In some embodiments, polymers, e.g., alginate polymers, are modifiedwith tetrazine or norbornene groups that can subsequently be covalentlycross-linked to form click-crosslinked hydrogels, e.g., Click alginatehydrogels. Click-crosslinked hydrogels are capable of encapsulatingcells, proteins, and other biological molecules with minimal damage. Forexample, the viability of a population of cells following encapsulationinto a hydrogel described herein in at least about 10%, e.g., at leastabout 10%, or about 20%, or about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95% orgreater. Viability of cells can be determined by standard methods in theart.

The cross-linking reaction has been previously shown by others to behighly specific, bio-orthogonal, and quick (see, e.g., Devaraj et al.Bioconjugate Chem. 19.12(2008):2297-2299; Karver et al. BioconjugateChem. 22.11(2011):2263-2270; and Alge et al. Biomacromol.14.4(2013):949-953), allowing for incorporation of cells with highpostencapsulation viability.

Exemplary polymers include but are not limited to alginate, chitosan,poly ethylene glycol (PEG), gelatin, hyaluronic acid, collagen,chondroitin, agarose, polyacrylamide, and heparin.

The first polymer and the second polymer may be formulated for specificapplications by controlling the molecular weight, rate of degradationand method of scaffold formation. Coupling reactions can be used tocovalently attach bioactive epitopes, such as the cell adhesion sequenceRGD to the polymer backbone (for example, see modified alginate below).Differences in hydrogel formulation control the kinetics of hydrogeldegradation. Release rates of pharmaceutical compositions, e.g., smallmolecules, morphogens, proteins, nucleic acids, or other bioactivesubstances, from alginate hydrogels is controlled by hydrogelformulation to present the pharmaceutical compositions in a spatiallyand temporally controlled manner. This controlled release eliminatessystemic side effects.

Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (Munits) and α L-guluronic acid (G units) monomers, which can vary inproportion and sequential distribution along the polymer chain.

In a preferred embodiment, the first polymer and the second polymer areindependently alginate, chitosan, or gelatin. In another embodiment, thefirst polymer and the second polymer are independently alginate. Inanother embodiment, the first polymer and the second polymer areindependently chitosan. In another embodiment, the first polymer and thesecond polymer are independently gelatin.

For example, the polymers (e.g., alginates) of the hydrogel are about50-100% crosslinked, e.g., at least about 50%, or about 60%, or about70%, or about 80%, or about 90%, or more crosslinked. The polymer (e.g.,alginate) can be oxidized, reduced, or neither, or a mixture thereof. Insome cases, oxidized polymers or partially oxidized polymers arebiodegradable. For example, hydrogels comprising oxidized or partiallyoxidized alginate are biodegradable.

Methods of preparing a hydrogel described herein are also provided bythe disclosure. For example, a schematic illustration a method forpreparing a hydrogel is shown in FIGS. 1 and 2.

The mechanical properties of the hydrogels described herein, e.g., clickalginate hydrogels or click gelatin hydrogels, can be tuned for a setpolymer concentrations via the degree of substitution of norbornene ortetrazine groups on the polymer chains or the ratio of norbomene totetrazine groups. This tuning allows for the creation of elastichydrogels with Young's moduli ranging from about 50 to about 50,000 Pa.

In some cases, the hydrogels described herein are strong. For example,upon compression or dehydration, the hydrogel maintains structuralintegrity, i.e., after compression or dehydration, the hydrogel regainsits shape after it is rehydrated or the shear forces of compression areremoved/relieved. The hydrogel also maintains structural integrity inthat it is flexible (i.e., not brittle) and does not break under sheerpressure.

The norbornene modified polymers (e.g., norbornene modified alginate)can also be modified with thiol-containing molecules or proteins viathiol-ene photochemistry either before or after crosslinking into ahydrogel. For example, the thiol-ene chemistry occurs beforecrosslinking. In such cases, the thiol reacts with all of the norbornenefunctional groups on the polymer(s). In other cases, there remainunreacted norbornene groups on the polymer(s) after reaction with thethiol. The reactivity can be controlled by varying the amount of thiolcontaining compound and the amount of polymer containing norbornenegroups (e.g., incubating a smaller number of moles of thiol with alarger number of moles of polymer containing norbornene. Alternatively,reactivity can be controlled by varying the degree of substitution ofthe polymer(s) with norbornene groups and, e.g., incubating a smallernumber of moles of thiol with a larger number of moles of totalnorbornene groups. In such a way, unreacted norbornene groups on thepolymer after the thiol-ene chemical reaction are available to reactwith tetrazine in the crosslinking reaction. In some examples, the thioldoes not react with the crosslinked norbornene-tetrazine product aftergelation.

As used herein, the terms “tetrazine” and “tetrazine moiety” includemolecules that comprise 1,2,4,5-tetrazine substituted with suitablespacer for linking to the polymer (e.g., alkylamines like methylamine orpentylamine), and optionally further substituted with one or moresubstituents at any available position. Exemplary tetrazine moietiessuitable for the compositions and methods of the disclosure include, butare not limited to, the structures shown below (see, e.g., Karver et al.Bioconjugate Chem. 22(2011):2263-2270, and WO 2014/065860, bothincorporated herein by reference):

As used herein, the terms “norbornene” and “norbomene moieties” includebut are not limited to norbomadiene and norbornene groups furthercomprising suitable spacer for linking to the polymer (e.g., alkylamineslike methylamine or pentylamine), and optionally further substitutedwith one or more substituents at any available position. Such moietiesinclude, for example, norbomene-5-methylamine andnorbomadienemethylamine.

A variety of polymers suitable for the click conjugation of thedisclosure allow for a drug delivery platform that can be used toconfigured for specific drug delivery goals through the materialspecific characteristics given herein. Exemplary applications includeuse as a dermal filler, in drug delivery, as a wound dressing, forpostsurgical adhesion prevention, and for repair and/or regenerativemedical applications such as cell therapy (e.g., immunoisolated celltherapy), gene therapy, tissue engineering, immunotherapy.

Finally, the hydrogels described herein are defect resistant, i.e., thedurable gel is not prone to development of tears. But even if a defectarises, the gel maintains its toughness and does not fail.

An advantage of the hydrogels described herein is that they arebiocompatible to cells (e.g., show lack of degradation and noinflammation in cells) over long periods of time, e.g., 3 days, 7 days,14 days, 28 days 56 days, 112 days, or 224 days.

The biocompatible hydrogels described herein offer significantadvantages, particularly in medical applications. For example, drugdelivery hydrogels or cell delivery hydrogels that are used for musclegeneration or regeneration are subject to application ofenergy/stresses. Because the hydrogels described herein are moremechanically robust, more durable, and are characterized by a higherfracture resistance compared to prior hydrogels, they are more suitablefor such applications involving muscle tissue. Other applications arealso improved with the use of the tough hydrogels. For example,materials used in surgical procedures (e.g., wraps, meshes), cartilagereplacement, joint replacement, orthopedic/orthochondral defect repair(e.g., bone or cartilage fillers), spinal procedures (e.g., nuclearpropulsus spinal surgery), ophthamological uses (e.g., optically-clear,flexible, durable lenses, contact lens or implantable lens), as well asnon-medical uses (e.g., fillers in cosmetic surgical procedures).

In addition to clinical uses such as tissue repair and replacement, thehydrogels are also useful in non-medical settings, such as infabrication of soft robotics that swim, crawl, fly, or squeeze throughsmall spaces without breaking. The hydrogels are also useful to makeactuators. Other examples include artificial muscles, tunable lenses,actuators and skins for soft robotics, encapsulate protecting layers,stretchable membranes for dielectric actuator, loud speaker membranes,multilayer systems, fiber reinforced tough hydrogel, particle reinforcedtough gel as well as durable filtration systems.

Biological factors such as polynucleotides, polypeptides, or otheragents (e.g., antigens) are purified and/or isolated. Specifically, asused herein, an “isolated” or “purified” nucleic acid molecule,polynucleotide, polypeptide, or protein, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or chemical precursors or other chemicals when chemicallysynthesized. Purified compounds are at least 60% by weight (dry weight)the compound of interest. The preparations herein can also be at least75%, more preferably at least about 90%, and most preferably at leastabout 99%, by weight the compound of interest. For example, a purifiedcompound is one that is at least about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 98%, about 99%, or about 100% (w/w) ofthe desired compound by weight. Purity is measured by any appropriatestandard method, for example, by column chromatography, thin layerchromatography, or high-performance liquid chromatography (HPLC)analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA)or deoxyribonucleic acid (DNA)) is free of the genes or sequences thatflank it in its naturally-occurring state. Purified also defines adegree of sterility that is safe for administration to a human subject,e.g., lacking infectious or toxic agents.

Similarly, by “substantially pure” is meant that a nucleotide,polypeptide, or other compound has been separated from the componentsthat naturally accompany it. Typically, the nucleotides and polypeptidesare substantially pure when they are at least about 60%, about 70%,about 80%, about 90%, about 95%, about 99%, or even 100%, by weight,free from the proteins and naturally-occurring organic molecules withthey are naturally associated. Examples include synthesized compounds,recombinant compounds (e.g., peptides, proteins, nucleic acids) orpurified compounds, such as purified by standard procedures includingchromatographic methods.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term covers, for example:(a) a DNA which is part of a naturally occurring genomic DNA molecule,but is not flanked by both of the nucleic acid sequences that flank thatpart of the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner, such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybridgene, i.e., a gene encoding a fusion protein. Isolated nucleicacid molecules according to the present disclosure further includemolecules produced synthetically, as well as any nucleic acids that havebeen altered chemically and/or that have modified backbones.

A “deployment signal” is a composition such as protein, peptide, ornucleic acid. For example, cells migrating into the hydrogel onlyencounter the deployment signal once they have entered the hydrogel. Insome cases, the deployment signal is a nucleic acid molecule, e.g., aplasmid containing sequence encoding a protein that induces migration ofthe cell out of the hydrogel and into surrounding tissues. Thedeployment signal occurs when the cell encounters the plasmid in thehydrogel, the DNA becomes internalized in the cell (i.e., the cell istransfected), and the cell manufactures the gene product encoded by theDNA. In some cases, the molecule that signals deployment is an elementof the hydrogel and is released from the hydrogel in delayed manner(e.g., temporally or spatially) relative to exposure of the cell to therecruitment composition. Alternatively, the deployment signal is areduction in or absence of the recruitment composition. For example, arecruitment composition induces migration of cells into the hydrogel,and a reduction in the concentration or depletion, dissipation, ordiffusion of the recruitment composition from the hydrogel results inegress of cells out of the hydrogel. In this manner, immune cells suchas T cells, B cells, or dendritic cells (DCs) of an individual arerecruited into the hydrogel, primed and activated to mount an immuneresponse against an antigen-specific target. Optionally, an antigencorresponding to a target to which an immune response is desired isincorporated into or onto the hydrogel structure. Cytokines, such asgranulocyte macrophage colony stimulating factor (GM-CSF) are also acomponent of the hydrogel to amplify immune activation and/or inducemigration of the primed cells to lymph nodes. For example, vascularendothelial growth factor (VEGF) is useful to recruit angiogenic cells.

The disclosure will be further described in the following examples,which do not limit the scope of the disclosure described in the claims.

EXAMPLES Example 1: 3-(p-benzylamino)-1,2,4,5 tetrazine synthesis

50 mmol of 4-(aminomethyl)benzonitrile hydrochloride and 150 mmolformamidine acetate were mixed while adding 1 mol of anhydroushydrazine. The reaction was stirred at 80° C. for 45 minutes and thencooled to room temperature, followed by addition of 0.5 mol of sodiumnitrite in water. 10% HCl was then added dropwise to acidify thereaction to form 3-(p-benzylamino)-1,2,4,5-tetrazine. The oxidizedacidic crude mixture was then extracted with DCM. After discarding theorganic fractions, the aqueous layer was basified with NaHCO₃, andimmediately extracted again with DCM. The final product was thenrecovered by rotary evaporation, and purified by HPLC. All chemicalswere purchased from Sigma-Aldrich.

Example 2: Click Alginate Polymer Synthesis

Click alginate biopolymers were modified with either1-bicyclo[2.2.1]hept-5-en-2-ylmethanamine (Norbornene Methanamine;Matrix Scientific) or 3-(p-benzylamino)-1,2,4,5-tetrazine by firstallowing high molecular weight alginate, M_(w)=265 kDa (Protanol LF20/40; FMC Technologies) to dissolve in stirred buffer containing 0.1 MMES, 0.3 M NaCl, pH 6.5 at 0.5% w/v. Next, N-hydroxysuccinimide (NHS;Sigma-Aldrich) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC; Sigma-Aldrich) were added in 5× molar excess of thecarboxylic acid groups of alginate. Either norbornene or tetrazine wasthen added at 1 mmol per gram of alginate to make Alg-N or Alg-T,respectively. The coupling reaction was stirred at room temperature for24 hours, after which the reaction was quenched with hydroxylamine(Sigma-Aldrich) and dialyzed in 12-14 kDa MWCO dialysis tubing (SpectrumLabs) for 4 days against a decreasing salt gradient from 150 mM to 0 mMNaCl in diH₂O. The purified Alg-N and Alg-T polymers were treated withactivated charcoal, sterile filtered (0.22 μm), and freeze-dried. Thisresulted in purified Alg-N or Alg-T polymers with a 5% degree ofsubstitution of the available carboxylic acid groups of alginate.

Example 3: Preparation and Characterization of Click Alginate Hydrogels

Click alginate hydrogels were prepared by first separately dissolvingfreeze-dried Alg-N and Alg-T polymers to final desired concentration(2-4% w/v) in Dulbecco's Modified Eagle Medium (DMEM; Gibco). Forgelation kinetics measurements, Alg-N and Alg-T polymer solutions weremixed at a desired ratio (i.e., 0.5-4:1 N:T) and directly pipetted ontothe bottom plate of a TA Instruments ARG2 rheometer equipped with 8 mmflat upper plate geometry. A Peltier cooler was used to control thetemperature for temperature dependent experiments, and mineral oil wasapplied to the gel periphery to prevent the hydrogel from drying duringtesting. Hydrogel samples were subjected to 1% strain at 1 Hz, and thestorage and loss moduli (G′ and G″) were monitored for 4 hours. ForYoung's modulus measurements click alginate hydrogels were formed undersiliconized glass plates (Sigmacote; Sigma-Aldrich) with 2 mm spacers.After 2 hours of crosslinking at room temperature, cylindrical diskswere punched using an 8 mm biopsy punch, transferred to DMEM, andswollen to equilibrium for 24 hours at 37° C. Swollen hydrogel sampledimensions were measured using calipers for volumetric swelling ratiomeasurements, and then subjected to unconfined compression testing (1mm/min) using a 10 N load cell with no preload (Instron Model 3342). TheYoung's modulus, E, was calculated as the slope of the linear portion(first 10%) of the stress vs. strain curves.

To prepare click alginate polymers, norbornene or tetrazine groups wereintroduced to high molecular weight alginate biopolymers usingconventional carbodiimide chemistry (FIG. 1). The degree of substitutionof norbornene or tetrazine groups onto purified click alginate polymerswas determined from ¹H NMR spectra. A 5% degree of substitution ofnorbornene (Alg-N) or tetrazine (Alg-T) on alginate carboxyl groups wasobtained using this method, and these batches of click alginate polymerswere used for all subsequent experiments.

To form click alginate hydrogels, Alg-N and Alg-T polymer solutions wereprepared separately and mixed together to gel. Upon mixing of the twoclick alginate polymers, a stable gel was formed via an inverse electrondemand Diels-Alder reaction between the two polymers, which releasesnitrogen gas (FIG. 2). The nitrogen gas evolved from the crosslinkingreaction does lead to the formation of a few small bubbles within thehydrogel. A stable gel was formed within 1 hour at 25° C. (FIG. 3A),though the gelation kinetics could be tuned by varying the temperatureor initial degree of substitution of the click alginate polymers (datanot shown). The gelation kinetics at 25° C. are favorable because itallows the user to easily achieve a well-mixed polymer formulationbefore gelation, a common challenge with other alginate hydrogelcrosslinking methods.

The mechanical properties of the extracellular matrix have been shown toaffect cell fate and function in 2D and 3D environments. In order totune mechanical properties over a wide range, click alginate polymerswere mixed at different ratios of Alg-N and Alg-T (N:T ratio) for agiven polymer concentration between 2 and 4% w/v. These click alginatehydrogel samples were subjected to unconfined compression testsresulting in a compressive Young's modulus that predictably increasedwith increasing polymer concentration, and decreased as the ratiobetween the polymers deviated from the stoichiometrically balanced N:Tratio of 1 (FIG. 3B). The ability to tune the mechanical properties ofthe resulting gel over a large range by simply changing the ratio of thetwo polymers allows control over gel stiffness while keeping otherparameters such as polymer concentration, and ligand density constantwhich may be useful for studies of mechanobiology.

The swelling ratio of hydrogel systems can affect mechanical properties,mass transport, and the presentation of ligands on the gel surface. Toinvestigate how volumetric swelling would change at different polymerconcentrations and N:T ratios, click alginate hydrogels were made aspreviously described and allowed to swell for 24 hours at 37° C. Theswollen volume was measured and compared to the casted volume (FIG. 3C).For a given polymer concentration, the volumetric swelling ratioincreased as the N:T ratio deviated from 1, demonstrating an inverserelationship between mechanical properties and swelling ratio asexpected. While the N:T ratio has a significant effect on the swellingratio, the polymer concentration does not have a significant effect,indicating that the swelling ratio of click alginate is dominated bycrosslink density rather than polymer concentration (data not shown).

Example 4: Viability of Cells Encapsulated in Click-CrosslinkedHydrogels

NIH 3T3 (ATCC) cells were transduced with lentivirus produced from anEGFP-containing lentiviral vector (pLCAG EGFP, Inder Verma lab, Addgeneplasmid 14857) and were selected for 7 days in 1 μg/mL puromycindihydrochloride (EMD Millipore). EGFP-expressing 3T3 fibroblast cellswere cultured in DMEM supplemented with 10% (v/v) fetal calf serum, 100U/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37° C., in a 5%CO₂ environment. Cells were passaged approximately twice per week.

For cell adhesion studies, slabs of click alginate hydrogels weremodified with cell adhesion peptides as described above. 6 mm disks werepunched, placed in DMEM, washed several times, and swollen for 4 hoursprior to seeding with cells at 5×10⁴ cells/mL at a depth ofapproximately 1 mm above the surface of the gel. Cells were given 24hours to adhere and spread and then visualized via EGFP fluorescenceusing an epifluorescence microscope. EGFP images were used to quantifytotal cell area using ImageJ software. After 3 days of culture, cellswere fixed and stained using Alexa Fluor 594 phalloidin (MolecularProbes) and Hoescht 33342 (Molecular Probes) to visualize F-actinfilaments and nuclei respectively. To visualize cell death, gels wereincubated for 20 minutes with a 4 μM ethidium homodimer-1 (MolecularProbes) solution in Hanks Buffered Saline Solution (HBSS) and imagedusing an epifluorescence microscope.

For cell encapsulation studies, Alg-N polymers were modified to haveapproximately 20 cell adhesive GGGGRGDSP peptides (Peptide2.0) peralginate chain as previously described. 600 μm thick click alginatehydrogels at 2% w/v, N:T=1, were then made containing cells at 3×10⁶cells/mL. Ionically crosslinked hydrogels were similarly prepared at 2%w/v using the same cell density and backbone RGD modified Alg-Npolymers. A CaSO₄ slurry (0.21 g CaSO₄/mL ddH₂O) at a finalconcentration of 2% w/v was used to crosslink the ionically crosslinkedhydrogel samples so as to match the mechanical properties of the twosubstrates as closely as possible. To minimize the time in which cellsdid not have access to culture media, gels were allowed to crosslink atroom temperature for 1 hour, after which 6 mm disks were punched andplaced in culture medium where the crosslinking reaction was expected toproceed to completion.

Cells were retrieved from alginate hydrogels by digestion in a 5 U/mLalginate lyase (Sigma-Aldrich) solution in HBSS for 20 minutes. Forviability testing, cells were stained with a Muse Count and ViabilityKit and tested on a Muse Cell Analyzer (EMD Millipore). To assess totalcell metabolic activity, gels were transferred to wells containing 10%AlamarBlue (AbD Serotec) in cell culture medium and incubated for 4hours. The reduction of AlamarBlue was assessed according to themanufacturer's instructions.

3T3 cells were encapsulated in hydrogels generated from Alg-N(alginate-norbomene) and Alg-T (alginate-tetrazine) that had low versushigh degrees of substitution at varying polymer concentrations (e.g.,1.5% or 3.0% w/v; i.e., 1.5 g alginate per 100 mL DMEM or 3 g alginateper 100 mL DMEM). Cells were stained with a commercial Live-Dead kitfrom Life Technologies. Degree of substitution is measured by nuclearmagnetic resonance (NMR), comparing the integration of peaks of alginateprotons to peaks of either alkene protons on norbomene, or tetrazineprotons. A quantitative measure of degree of substitution can beobtained from this using the ratio of norbomene/tetrazine protons to thealginate protons to calculate a degree of substitution.

Cell viability and metabolic activity of cells encapsulated in clickalginate hydrogels were also investigated over a 3 day culture period;ionically crosslinked hydrogels were used for comparison in thesestudies. Representative images of encapsulated cells stained withethidium homodimer-1 show minimal cell death in both click and ionicallycrosslinked gels 4 hours and 3 days after encapsulation (FIG. 5A).Quantification revealed that click alginate hydrogels resulted insignificantly higher viability of encapsulated 3T3 cells bothimmediately after encapsulation (93±1% vs. 87±2%) and after 3 days ofculture (84±2% vs. 79±4%) (FIG. 5B). It should be noted that a loss inmeasured cell viability may occur during the cell retrieval process byenzymatic digestion of the hydrogels. The overall metabolic activity ofthe cells encapsulated in the different hydrogels was also analyzed, andnoted to increase over the 3 day culture period for both hydrogelcrosslinking chemistries (results not shown).

Example 5: Post-Gelation Thiol-Ene Photoreaction of Click-CrosslinkedHydrogels

Click alginate hydrogels were made as described above (2% w/v, N:T=2)and then a cell adhesive CGGGGRGDSP peptide (Peptide2.0) solution at 0.2or 2 mM containing 0.5% w/v photoinitiator (Irgacure 2959;Sigma-Aldrich) was pipetted on top and the gel was covered with a glasscoverslip. Gels were irradiated at 365 nm for 60 seconds at 10 mW/cm².The gels were washed several times with DMEM to remove excessphotoinitiator and unreacted peptide and swollen to equilibrium at 37°C. before seeding with cells.

Thiol-containing molecules were grafted onto unreacted norbornenes inpre-formed click alginate hydrogels using a photoinitiated thiol-enereaction (FIG. 5A). Gels with N:T=2 were used to ensure unreactednorbornenes were available to react after the initial gelation. RGDpeptide solutions at high (2 mM) or low (0.2 mM) concentration werereacted onto the surface of these click alginate hydrogels and then gelswere seeded with NIH 3T3 fibroblasts expressing a cytosolic fluorescentmarker (EGFP). 3T3 cells readily adhered and spread on gels modifiedwith RGD, while very few cells were able to attach or elongate oncontrol gels with no RGD (FIG. 5B). Cells on click alginate hydrogelspresenting RGD were able to form branched interconnected networks, witha significant RGD density-dependent 2-3 fold increase in surfacecoverage over the 3 day culture, while unmodified click alginate gelswere observed to be non-cell-adhesive and showed a decrease in surfacecoverage by cells over time (FIG. 5C). After 3 days in culture, cellsalso showed an increase in spreading and actin stress fiber formationwith higher RGD concentration (FIG. 5D). Additionally, the highviability of cells after 3 days of culture demonstrated thecytocompatibility of the click alginate hydrogels for 2D cell culture(FIG. 5E).

Example 6: In Vivo Hydrogel Inflammatory Response

Ultrapure alginate with low endotoxin levels (MVG alginate, ProNovaBiomedical AS) was modified as described above with norbornene andtetrazine and subsequently prepared at 2% w/v in DMEM afterpurification. Click alginate hydrogels were prepared by mixing ultrapureAlg-N and Alg-T polymers with N:T=1 by connecting two syringes with aluer lock. 15 minutes after mixing, 50 μL of click alginate hydrogel wasinjected subcutaneously through an 18 G needle. For ionic hydrogelsamples, a 2% w/v ultrapure alginate solution was prepared in DMEM andsimilarly mixed in a syringe with a CaSO₄ slurry at a finalconcentration of 2%. 50 μL of the ionically crosslinked gel was alsoinjected subcutaneously in the same mice. Both gel samples wereretrieved along with the surrounding skin after 1 week, 1 month, and 2months of injection and fixed overnight in 10% neutral buffered formalinsolution (Sigma-Aldrich). Samples were embedded in paraffin, sectioned,and stained with hematoxylin and eosin.

The inflammatory response to the injection of click alginate hydrogelsin vivo was investigated next. Click crosslinked and ionicallycrosslinked alginate hydrogels were injected subcutaneously andretrieved after 1 week, 1 month, and 2 months. The gelation kinetics ofclick alginate hydrogels allows them to be mixed and readily injected,in a similar manner to ionically crosslinked hydrogels. A thin fibrouscapsule was found to surround both types of gels 1 week after injection.H&E staining revealed a very thin capsule of collagen and fibroblastssurrounding the material throughout the duration of the study withminimal inflammation (FIG. 6A). At 1 month, the ionically crosslinkedgels were seen to lose structural integrity and allowed for infiltrationof fibroblasts and immune cells into the gel, while the clickcrosslinked samples showed no evidence of breakdown nor cellinfiltration into the material for up to 2 months (see FIG. 6B), andmaintained a thin layer of fibroblasts surrounding the gel.

Example 7: Preparation of Click Chitosan Hydrogels

4-(1,2,4,5-Tetrazin-3-yl)benzoic acid was synthesized according topreviously published methods and purified by preparatory reverse-phaseHPLC. 5-norbornene-2-carboxylic acid was purchased from Sigma Aldrich.Tetrazine (Tz) and Norbomene (Nb) acids are conjugated to Chitosanamines by solubilizing Tz or Nb in DMSO, adding EDC and NHS. Chitosan,solubilized overnight in 0.1 M HCl and then pH adjusted to ˜6 throughaddition of 1M MES Buffer pH 6.5, is then added dropwise to the Tz/NbDMSO solution. The reaction is allowed to react overnight at roomtemperature with stirring. The resulting solution is dialyzed againstwater and then lyophilized. The resulting materials are resuspended inphysiologically relevant pH buffered solution or WFI are reacted toproduce hydrogels.

Chitosan-Nb, Chitosan-Tz, and unmodified Chitosan polymers weredissolved in deuterium oxide (Sigma-Aldrich) at 1.5% w/v. 1H-NMR spectrawere obtain on a 400 MHz NMR spectrometer (Varian). Degree ofsubstitution is calculated by comparing the integral of the chitosanbackbone proton peak δ 3.0 with either the alkene proton peaks ofnorbomene at δ 6.2-5.9 or with peak of tetrazine at δ 10.4 (s, 1H).

Example 8: Preparation of Click Gelatin Hydrogels

Similarly to alginate and chitosan hydrogels, gelatin was modified toprepare gelatin-tetrazine (Gel-Tz) and gelatin-norbornene (Gel-Nb).Gel-Tz and Gel-Nb polymers are mixed together to create a covalentlycrosslinked click gelatin hydrogel network, with the loss of N₂. (FIG.7A). Storage modulus (G′) time sweeps of click gelatin hydrogels at 5and 10% w/v and N:T=1 show that plateau modulus were reached within 45min for both materials, with inset showing time to 50% of plateaumodulus. FIGS. 8A and 8B show 3T3 fibroblast adhesion, spreading, andproliferation on click gelatin hydrogels. Intrinsic cell adhesivepeptide sequences of gelatin were preserved as cells were seen toreadily adhere and spread very quickly on the surface of the gel (2Dassay). F-actin and nuclei staining using Phalloidin and Hoescht of 3T3fibroblast adhesion, spreading, and proliferation in FIG. 8B showsstrong pull on the underlying matrix by the cells.

FIG. 9 shows that 3T3 fibroblasts retained high viability after 3Dencapsulation in click crosslinked gelatin with an increase in metabolicactivity over a three day culture period. Encapsulated 3T3 fibroblastsrapidly assumed a spread morphology within click crosslinked gelatinafter one day (cell length: 80±6 m), a phenotype that was inhibited inthe presence of the broad-spectrum matrixmetalloproteinase(MMP)-inhibitor marimastat (cell length: 16±2 μm),suggesting 3D cell spreading in click crosslinked gelatin was dependenton MMP-mediated degradation of the gelatin matrix.

While the disclosure has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the disclosure, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this disclosure has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the disclosureencompassed by the appended claims.

We claim:
 1. An injectable hydrogel comprising a first polymer and asecond polymer, wherein the first polymer and the second polymer are thesame polymer and are both alginate, and wherein the first polymer isconnected to the second polymer by linkers of formula (A):

wherein bond

is a single or a double bond; R¹ is —C₀-C₆ alkyl-NH—, —C₀-C₆ alkyl —O—,or —C₀-C₃alkyl-C(O)—; R² is a bond, aryl, or heteroaryl, wherein aryland heteroaryl are optionally substituted with halogen, hydroxy, C₁-C₆alkyl, C₁-C₆ alkoxy, (C₁-C₆ alkyl)amino, or di(C₁-C₆ alkyl)amino; R³ is—C₀-C₆ alkyl-NH—, —C₀-C₆ alkyl-O—, or —C₀-C₃alkyl-C(O)—; and R⁴ ishydrogen, C₁-C₆ alkyl, aryl, or heteroaryl, wherein aryl and heteroarylare optionally substituted with halogen, hydroxy, C₁-C₆ alkyl, C₁-C₆alkoxy, (C₁-C₆ alkyl)amino, or di(C₁-C₆ alkyl)amino; wherein the Young'smodulus of the hydrogel is about 500 Pa to about 50,000 Pa.
 2. Thehydrogel according to claim 1, wherein: bond

is a single bond; R¹ is —C₀-C₆ alkyl-NH—, or —C₀-C₃alkyl-C(O)—; R² is abond or aryl optionally substituted with halogen, hydroxy, C₁-C₆ alkyl,C₁-C₆ alkoxy, (C₁-C₆ alkyl)amino, or di(C₁-C₆ alkyl)amino; R³ is —CO—C₆alkyl-NH—, or —C₀-C₃alkyl-C(O)—; and R⁴ is hydrogen, C₁-C₆ alkyl, orheteroaryl, wherein heteroaryl is optionally substituted with halogen,hydroxy, C₁-C₆ alkyl, C₁-C₆ alkoxy, (C₁-C₆ alkyl)amino, or di(C₁-C₆alkyl)amino.
 3. The hydrogel according to claim 2, wherein R¹ and R³ areboth -methyl-NH—; or R¹ and R³ are both —C(O)—.
 4. The hydrogelaccording to claim 1, wherein the linkers of formula (A) are selectedfrom the group consisting of formula (I):

formula (II):

and formula (III):


5. The hydrogel of claim 1, wherein the first polymer or the secondpolymer further comprises a cell adhesive peptide.
 6. The hydrogel ofclaim 5, wherein the cell adhesive peptide (a) comprises the amino acidsequence RGD; (b) comprises the amino acid sequence RGDC; and/or (c) iscovalently linked to the polymer via a thiol-ene reaction.
 7. Thehydrogel of claim 1, wherein the hydrogel further comprises andencapsulates a cell, a biological factor, and/or a small molecule. 8.The hydrogel of claim 7, wherein the cell is a mammalian cell; whereinthe biological factor is a protein, nucleic acid, lipid, orcarbohydrate; and/or wherein the protein is a growth factor or fragmentthereof or an antibody or fragment thereof.
 9. A method of delivering acell, a biological factor and/or a small molecule to a subject, themethod comprising administering to the subject the hydrogel of claim 7,wherein after administration of the hydrogel, the cell, the biologicalfactor and/or the small molecule is released from the hydrogel into asurrounding tissue of the subject, thereby delivering the cell, thebiological factor and/or the small molecule to the subject.
 10. A methodfor preparing a hydrogel of claim 1, the method comprising a) providinga first polymer comprising a tetrazine moiety and a second polymercomprising a norbornene moiety; b) contacting the second polymer withthe first polymer to form the hydrogel of claim
 1. 11. The method ofclaim 10, wherein (i) each molecule of the first polymer comprises1-50,000 tetrazine moieties; (ii) each molecule of the second polymercomprises 1-50,000 norbornene moieties; and/or (iii) step b) comprisescontacting a second polymer with a first polymer at a ratio of about1:10 to about 10:1.
 12. The method of claim 10, wherein the firstpolymer comprising a tetrazine moiety is generated by reacting the firstpolymer with a) benzyl amine tetrazine, benzyl alcohol tetrazine, orbenzoic acid tetrazine, and b) a coupling agent.
 13. The method of claim10, wherein the second polymer comprising a norbornene moiety isgenerated by reacting the second polymer with a) norbornene methanamine,norbornene methanol, or norbornene carboxylic acid, and b) a couplingagent.
 14. A method of regenerating a tissue in a subject in needthereof, the method comprising contacting the tissue with the hydrogelof claim 1.